ISr7874(PartII)- 1975 Indian Standard ( Reaffirmed 2004 ) METHODS OF TESTS FOR ANIMAL FEEDS AND FEEI%NG STUFFS PART II MINERALS AND TRACE ELEMENTS ( Second Reprint DECEMBER 1992 ) UDC 636.085.12 @ Cofiyright 1976 BUREAU OF INDIAN STANDARDS MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARC3 NEW DELHI 110002 Gr 5 April 1976 IS : 7874 ( Part II ) - 1975 Indian Standard METHODS OF TESTS FOR' ANIMAL FEEDS AND FEEDING STUFFS PART II MINERALS Feeds Sectional AND TRACE ELEMENTS AFDC 15 Animal Chairman DRB.K.SONI Members AGRICULTURAL ADVISER TO Committee, Representing Indian Council of Agricultural Research, New Delhi M A R K E T IN G THE GOVERNMENT 0~ INDIA SHRI S. JAYARAMAN(Alternate ) Godrej Soaps Private Limited, Bombay DR S. L. ANAOKAR DR S. P. ARORA National Dairy Research Institute ( ICAR), Karnal DR B. N. GUPTA ( Alternnte) U. P. College of Veterinary Science & Animal DR C.S.BARSAUL Husbandry, Mathura DR D. C. JOSHI ( Alternate ) Directorate of Military Farms, Army Headquarters BRIG R. C. DATTA &RI R. K. TRIPATIII ( Alternate ) Directorate of Sugar and Vanaspati (Ministry of DIRECTOK( VANASPATI) Agriculture & Irrigation), New Delhi DEPUTY DIRECTOR ( VANASPATI) ( Alternate ) The Compound Livestock Feeds Manufacturers' DR N. S. DRONAWAT Association of India, Bombay SHRI V. VIRMANI (Alternate ) Voltas Limited, Bombay SHRI R. L. GAJWANI SHRI K. R. FRANCIS ( Alternate) Animal Husbandry Commissioner, Ministry of DR A. N. GHOSH Agriculture & Irrigation, New Delhi SHRI S. S. CHIIIBBAR ( Alternate ) HEAD, ANIMALNUTRITIONDIVISION Indian Veterinary Research Institute ( ICAR ,. ). Izatnagar . Punjab Agricultural University, Ludhiana DR J. S. ICFHPONANI DR L. S. HUNDAL ( Alternate ) ( Continued on page 2 ) @ BUREAU OF Copyright INDIAN 1976 Directorate of Marketing and Inspection ( Ministry of Agriculture & Irrigation ), Faridabad STANDARDS This publication is protected under the Indian Copyright Act (-XIV of 1957) and reproduction in whole or in part by any means except with written permission of the publisher shall be deemed to be an infringement of copyright under the said Act. iS:7874(PartII)-1975 M6mbcrs DR M. G. JACKSON Shnr N. S. MAINI RC/WcsCnting Govind Ballabh Pant University of Agriculture Sr Technology, Pantnagar ( Dist Nainital ) Department of Agriculture, Ministry of Agriculture & Irrigation, New Delhi Development Commissioner, Maharashtra, Bombay Government of SHRI S. S. CHAUHAN ( Al&male ) Dairy DR V. M. PATIL DR B. SAHAI ( &t6mUt6 ) DR D. V. RANGNEKAR Bhartiya Agro-Industries Foundation, Uruli-Kanchan ( Dist Pune ) .`!;a~ Wallace & Company Limited, Madras DR D. V. R. PRAKA.QI RAO SIxR1B. LAKsHMANAN ( Alfemate ) All India Association of Poultry Industry, Bombay DR A. P. SACHDEV DR G. F. MITHUJI'( Altcrnatc) College of Veterinary Science, Assam Agricultural DR A. SAIKIA University, Gauhati DR P. K. SxaRmA (Alternate ) Roller Flour Millers' Federation of India, New DR S. K. SARKAR Delhi SHRIS. M. MAZUMDAR ( Alternate ) DR (SYT) SAROJINI CHANDRAKaira District Co-operative Milk Producers' Union Limited, Anand SHEKHARAN DR A. S. DAVE ( Alternate ) DR N. SATPATHY The Tata Oil Mills C;ompany Limited, Bombay DR K. C. DANDONA (Alternate) Hindustan Lever Limited, Bombay ~DR H. 6. SAXENA Da S. YAMDAGNI I Alternate ) DR~K. C. SEN ' ' In personal capacity ( 403, Jodhpur Par!;, Calcutta 31) Directorate General of Technical Development SHRI K. N. R. SHARMA b!h;istry of Industries & Civil Supplies ), New DR P.C. SHUKLA SHRI G.V. S~RUR SHRI R. V. IQUR ( Alternate ) SHRI SURESH CHANDRA SHRIT.S.TAMBOLIA Institute of Agriculture, Anand All India Cottonseed Crushers' Association, General Molasses Co (P) ( Ghyziabad ) The marashtra Agro-Industries Corporation Limited, Bombay Ltd, Bombay Sahibabad Development DR D. N. RANADE ( Allematc ) Solvent Extractors' Association of India, Bombay SHRI L. J. TANNA SHRI M. K.RAHmAN (.&?matc, ) Jawaia kiour Mills, New Delhi SHRS VINEET V~RMANX SHRIANANDVIRMANI (Akmatc) Director Geheral, IS1 ( Eu-oficio Mcmbrr ) SHRI T. PURNANANDAM, Deputy Director ( Agri & Food ) secretary SHRX G. S. VILKHV Deputy Director ( Agri & Food ), IS1 ( Conh46d on page 20 ) 2 IS:7874(PartII)-1975 Indian Standard METHODS OF TESTS FOR ANIMAL FEEDS AND FEEDING STUFFS PART II MINERALS AND TRACE ELEMENTS 0. FOREWORD 0.1 This Indian Standard ( Part II ) was adopted by the Indian Standards Institution on 28 November 1975, after the draft finalized by the Animal Feeds Sectional Committee had been approved by the Agricultural and Food Products Division Council. 0.2 The importance of adoption of standard and uniform methods of analysis for quality control purposes need no emphasis. Such methods not only help in reducing divergence in the analytical results but also ensure and enable a proper comparison and correct interpretation of the test results. 0.3 During the past ~decade a large number of Indian Standards covering a wide range of animal feeds, feeding stuffs and feed suppleme,rts have been issued and many more are envisaged to be formulated. All these standards It was, therefore, considered include methods of tests that are common. desirable to have all test methods in a consolidated form as applicable to the whole range of animal feeds, feeding stuffs and supplements. It was felt that such a step would not only facilitate easy reference but also prevent undue repetition. Accordingly, this standard is being issued. 0.4 It is intended to issue this standard in three parts. Pari I covers general methods such as determination of moisture, crude protein, crude fat, crude fibre, total ash, acid insoluble ash, castor husk and MAHUA cake. This standard will cover methods for determining minerals and trace elements, and Part III covers the microbiological methods. 0.5 In reporting the result of a test or analysis made in accordance with this standard, if the final value, observed or calculated, is to be rounded off, it shall be done in accordance with IS : Z-1960*. 1. SCOPE 1.1 This standard ( Part II ) prescribes methods for determination of Gnerals and trace elements in animal feeds, feeding stuffs and feed supplements. *Rules for rounding off numerical values ( revireii ). 3 IS : 7874 ( Part II ) - 1975 2. QUALITY OF REAGENTS and distilled water ( see 2.1 Unless specified otherwise, pure chemicals IS : 1070-1960" ) shall be employed in tests. NOTE- ` Pure chemicals ' shall mean chemicals that do not contain impurities which affect the test results. 3. PREPARATION OF THE SAMPLE 3.1 If necessary, the test sample should be ground so that it passes through 850-micron IS Sieve (see IS : 460-19621` ). Alternatively, ASTM Sieve 20 or BS Sieve 18 or Tyler Sieve 20 may be used. 4. DETERMINATION OF SALT (CHLORINE AS SODIUM CHLORIDE 4.1 ) Reagents 60 g 4.1.1 Ferric Sulphate Solution -Dissolve [ Fe, ( SO4 )s ] in one litre of water. of ferric sulphate 4.1.2 Ammonium @droxide Solution - prepared by mixing ammonium hydroxide with 12 volumes of water. 4.1.3 Concentrated Nitric Acid - one volume of ). ( m/v) r.d. 1.42 ( see IS : 264-1968$ 4.1.4 Ferric &&hate Indicator Solution -Prepare as 25 percent solution. Filter and add equal volume of nitric acid. 4.1.5 4.1.6 Standard Silrrer Nitrate Solution 0.1 N. 0.1 N. Standard Potassium Thiocyanate Solution - 4.2 Procedure 4.2.1 Weigh accurately about 1 g of the dried material .and transfer to a 250-ml graduated flask. Add 50 ml of the ferric sulphate solution with a pipette, gently swirling the flask. Add 100 ml of the ammonium hydroxide solution to bring it to the mark. Swirl the flask enough to ensure thorough mixing but avoid vigorous shaking. Allow to settle for Filter through 1 l-cm Whatman filter paper No. 41 or its 10 minutes. equivalent. Transfer from the filtrate, an aliquot of 25 ml to a 25O-ml beaker. Add 10 ml of nitric acid and 10 ml of the ferric sulpbate indiThen add with constant stirring, known quantity of the cator solution. *Specification for water, distilled quality ( revised ). tspecification for test sieves ( revised ). fspecification for nitric acid (first revision ). 4 IS : 7874 ( Part II ) - 1975 Heat the solution to the standard silver nitrate solution in slight excess. boil and cool to room temperature and stir to coagulate the precipitate. Titrate the excess of silver nitrate with the standard potassium thiocyanate solution. The end point is indicated by the first appearance of red& !, tint that persists for 15 seconds. 4.3 Calculation 4.3.1 Chlorine as sodium chloride ( on moisturefree basis ), percent by mass = 5.845 ( AA', m( 100-M) silver nitrate solution, thiocyanatc solution, solution, the for R.YVz) where A = volume used, Jvl= normality in ml of the standard solution of the standard silver nitrate B = volume in ml of the standard potassium solution used up by the excess silver nitrate N, = normality m = mass in ( see 4.2.1 M = percent 5. DETERMINATION 5.1 Reagents of the standard g of the ) , and moisture dried potassium material thiocyanate taken content. OF CALCIUM 5.1 .l ffvdrochloric Acid( seeIS : 265-1962* ) diluted 25 ml of to 100 ml. concentrated hydrochloric acid .5.1.2 Methyl Red Indicator water. 5.1.3 5.1.4 5.1.5 5.1.6 5.1.7 1968#). Dissolve 0.15 g of methyl 50 percent ( v/v). red in 500 ml of Ammonium Hydroxide Solution - Dilute Ammonium Hydroxide Solution Ammonium Oxalate Solution Concentrated Sulphuric AcidStandard saturated. 2 percent ( v/v). r.d. 1.84 ( see IS : 266-1961-/- ). Solution 0.1 N ( see IS : 2316- Potassium Permanganate ( ,iYJt *Specification for hydrochloric acid ( revised ) . tSpecification for sulphuric acid ( rcviscd ). $Methods of preparation of standard solutions for calorimetric and volumetric anal@ Icuirh ). 5 IS : 7874 ( Part II ) - 1975 5;2 ~Procedure 5.2.1 Ashing and Extraction - Accurately weigh about.3 g of the material into a silica dish. Char carefully and continue the ashing in a muffle furnace at a temperature not above 450% until the ash is white or almost so. Cool the ash, moisten with a few millilitres of distilled water and add 3 to 5 ml of concentrated hydrochloric acid drop by drop. Evaporate to dryness on a water-bath and continue heating on the water-bath for one -hour to render silica insoluble. Moisten the residue with 20 ml distilled water and add about 2 to 3 ml of concentrated hydrochloric acid. Heat on a water-bath for a few minutes and filter through medium filter paper into a 250-ml volumetric flask. Wash the filter paper thoroughly with hot water, cool the filtrate and make it up to volume, shake thoroughly. 5.2.2 Transfer a 25-ml aliquot of the solution prepared as in 5.2.1 to a 400-ml beaker, dilute to about 100 ml with water and add two drops of methyl red indicator soltition. Add ammonium hydroxide solution dropwise till a brownish-orange colour is obtained ( PH 5% ). Add two drops of hydrochloric acid so that the colour of solution. is pink ( pH_2.5 to 3.0 ). Di!ute to about 150 ml, bring to the boil and add slowly, with constant stirring, 10 ml of hot ammonium oxalate solution. If the red colour of the solution changes to orange or yellow, add hydrochloric Leave overnight to acid dropwise until the colour again changes to pink. allow the precipitate to settle. Filter the supernatant liquid through ashless filter paper and wash the precipitate thoroughly with dilute ammonium hydroxide solution. Place the paper with the precipitate in the beaker in which precipitation was carried out and add a mixture of 125 ml Heat to 70 to 90°C and of water and 5 ml of concentrated sulphuric acid. titrate with the standard potassium permanganate solution until the first slight pink colour is obtained. 5.3 Calculation 5.3.1 Calcium percent where ( as Ca) ( on moisture-free by mass basis ), 2 000 A.% =??Q100-iI4M) A = volume in ml of the standard potassium permanganate solution required in the titration, N = normality of the standard potassium permanganate solution, m = mass in g of the material taken for the test ( see 5.2.1), and M = percent moisture content. 6. DETERMINATION OF PHOSPHORUS r.d. 1.42 ( see 1s : 264-1968* ). 6.1 Reagents 6.1.1 Concentrated Nitric Acid *Specification for nitric acid (Jirzt revision). 6 IS : 7874 ( Part II) - 1975 6.1.2 .Nitric Acid ( 1: 1) nitric acid and water. A mixture of equal volumes of concentrated 6.1.3 Ammonium Molybdate Stock Solution`Take 200 m of powdered ammonium molybdate In a stoppered graduated cylinder o? 1 000 ml capacity, add to it 800 ml of water and shake well for 25 minutes to dissolve the ammonium molybdate. Add gradually 25 percent ( m/v ) ammonium hydroxide solution till the solution is clear ( about 100 to 140 ml of ammoAvoid adding excess of ammonia. nium hydroxide may be required ). If necessary, filter the solution through Make up the volume to one litrc. a fluted filter paper and stock this solution. 6.1.4 6.1.5 6.1.6 6.1.7 Ntric Acid Solution -- 2 percent Nitrate Solution ( m/v ). ( m11r). Potassium 3 percent Standard Sodium l+droxide Sol&ion -- 0.1 N. 0.1 N. Standard .,Vitric Acid Solution - 6.1.8 Phenolphthalein Indicator Solution -- L)issolvc 0.1 g of phenolphthalein in 100 ml of 60 percent ( m/v) rectified spirit ( see IS : 323-1959* ). ~6.2 Procedure 6.2.1 Pwcijifation -Take a lo-ml aliquot of the prepared solution In a dry beaker, prepare ammonium ( .ree 5.2.1 ) in a 150-ml beaker. molybdate solution by pouring into it, quickly and sitnultaneously IO ml of the ammonium molybdate stock solution ( see 6.1.3 ) and 10 ml of concentrated nitric acid; or take 10 ml of concentrated nitric acid first in the beaker and into this pour quickly 10 ml of the ammonium molybdate stock Pour this freshly prepared solution, whirling the beaker during addition. clear liquid quickly into the beaker containing the aliquot and stir. NOTE-The temperature developed in the molybdate solution is sufftcient to precipitate all the phosphorus present in the aliquot. Under no circumstances the phosphomolybdate precipitate should be heated either on a water-bath or directly over a burner so as to avoid precipitation of molybdic anhydride. 6.2.2 Filtration and Washing - Allow the precipitate to stand overnight and then filter through a disc ofwhatman filter paper No. 42 in a Gooch crucible by suction or through a g-cm Whatman filter paper No. 42 over As far as possible only the supernatant liquid is an ordinary funnel. passed through the filter paper, retaining the precipitate in the beaker. When the supernatant liquid is decanted off, the precipitate is washed twice with dilute nitric acid and then with potassium nitrate solution until the washings are free from acid. If ordinary funnel and filter paper are used, freedom from acidity may be tested by collecting sufficient filtrate in a test*Specification for rectified spirit ( mixed ). 7 IS : 7874 ( Part II ) - 1975 tube to which a few drops of phenolphthalein indicator solution and one drop of the standard sodium hydroxide solution arc added. If the pink colour appears with one drop of the standard alkali, the pre,cipitate is free from acid. 6.2.3 Titration - Transfer the precipitate with the filter paper back to the beaker in which precipitation was carried out. When Gooch crucible is used for filtration, transfer the whole crucible along with the filter paper Add sufficient to the beaker in which precipitation was carried out. quantity of the standard sodium hydroxide solution from a burette just sufficient to dissolve the precipitate and then add 5 ml in excess. See that no yellow precipitate sticks to the filter paper. Note the total volume of the standard sodium hydroxide solution added. Add about 10 drops of phenolphthalein indicator solution and titrate the excess of alkali with the standard nitric acid. 6.3 Calculation 6.3.1 Phosphorus ( on moisture-free percent by mass basis ), 2: 336.75 ( A& m(lOO-M) l&V-,) - where A = volume in ml of the standard used ( see 6.2.3 ), J\fl = normality of the standard sodium hydroxide solution sodium hydroxide solution, used in to nitric acid B = volume in ml of the standard neutralize the excess alkali ( see 6.2.3 ), Jv, normality of the standard nitric acid, m = mass in g of the material and M = percent 7. DETERMINATKON moisture content. taken for the test ( ses 5.2.1. ), OF IRON 7.1 Apparatus 7.1 .l Heat Resistant 7.1.2 7.1.3 Cent$qe Photoelectric Glass Tube of 50 ml capacity, the isoamyl marked alcohol at 30 ml. phase. -suitable for clarifying 495 nm. Calorimeter - capable of measuring optical density at 7.2 Reagents 7.2.1 Distilled Water - redistilled. 8 IS t 7874 ( Part II ) - 1975 7.2.2 7.2.3 7.2.4 7.2.5 7.2.6 Concvntruted SulphuritAcidPerchloric Acid 60 percent r.d. 1.84 ( see IS : 266-1961* ). ( tn/m ) solution. ( m/m ). ( m/m ). ( m/m ). ( m/m ) solution in water Concentrated .Nitric Acid - 60 percent Ammonium HJjdroxide Solution Concentrated Hydrochloric Acid - 25 percent 35 percent 7.2.7 Hydrogen Peroxide Solution - 0.1 percent stored in a brown bottle in a refrigerator. 7.2.8 7.2.9 cyanate Isoarnyl Alcohol of boiling point Potassium Thiocyanate Solution -Dissolve ( KSCN ) in 100 ml of water. 129 to 132°C. 50 g of potassium thio- 7.2.10 Standard Iron Solution13issolve O-702 2 g of ferrous ammonium 6H>O] in 100 ml of water, add 5 ml of sulphate [ FeSOd( NH4 )$OI, concentrated sulphuric acid, warm slightly and add potassium permanganate solution (approximately 0.1 N ) drop by drop until the solution Make up the volume to one litre in a shows a slight pink colouration. graduated flask. Pipette 10 ml of this solution into a one-litre graduated flask, add 10 ml of hydrogen peroxide solution and make up the volume This solution contains 1 rug of iron per mill% tre. with water. 7.3 Procedure 7.3.1 Preparation of the Test Solution-Weigh accurately about 2.0 g of the material and transfer to a 200-ml Erlenmeyer flask. Add 2 ml of concentrated sulphuric acid, 3 ml of perchloric acid and 5 ml of concentrated nitric acid. Digest until a clear solution is obtained and white fumes Dilute with 10 ml of water and make up of sulphuric acid are evolved. the volume to 200 ml with water in a graduated flask. Preserve this solution for the determination of copper (see 9.3.1 ) and cobalt ( see 11.3.1 ). 7.3.2 Take a suitable aliquot of the test solution containing about 10 pg of iron and transfer to the heat resistant glass tube. Add ammonium hydroxide solution until the solution is just alkaline to phenolphthalein. Add 1 ml of concentrated hydrochloric acid and 1 ml of hydrogen peroxide solution and make up the volume in the tube to 30 ml with distilled water. Add 10 ml of isoamyl alcohol, accurately measured, and 2 ml of potassium thiocyanate solution, stopper the tube and shake for 20 seconds. Transfer enough of the isoamyl alcohol phase meant for colour measurement to the and centrifuge for 5 minutes at about 3 009 rev/min. centrifuge tubes, Measure the absorption of the solution in a suitable photo-electric calorimeter at 495 nm setting the reading of the blank at zero absorption. The blank is prepared simultaneously by using the same quantities *Specification for sulphuric acid ( revised). 9 IS : 7874 ( Part II ) - 1975 of acid employed in the digestion, the colour in the same size aliquot of the test solution. making up the volume and in the same manner and developing as in the case 7.3.3 Prepare a series ofstandards by treating aliquots of the standard iron solution (see 7.2.10) in the same manner as the test solution. From the absorption of the standard solutions, prepare a standard curve plotting absorption values against concentrations. From this curve, obtain the mass ofiron present in the test solution and calculate the quantity of iron present in 100 g of the material on moisture-free basis. 8. DETERMINATION OF IODINE ( as KI ) 8.1 Reagents 8.1 .l `Takadiastase 0.50 2 N. g of methyl orange in 8.1.2 Methyl Orange Indicator-Dissolve water and dilute to one litre. 8.1.3 Dilute Sulphuric Acid approximat.ely 8.1.4 Bromine Water - Saturated aqueous solution. Determine the appfbfimate concentration ( mg/ml) by adding ( from a burette ) a measured volume to a flask containing 5 ml of 10 percent potassium iodide solution, adding 5 ml of dilute sulphuric acid and titrating the liberated iodine with 0.1 N sodium thiosulphate solution. 8.1.5 8.1.6 8.1.7 Sodium Sulphite Solution Phenol Solution Sodium Solution Potassium Iodide Solution approximately approximately one percent ( m/v ). 10 percent 0.005 ( m/v ). N ( freshly ( m/v ). approximately Thiosulphate 1 percent 5 percent 8.1.8 Standard standardized ). 8.1.9 Starch Solution - ( freshly prepared ) ( m/v ). chloride in Solution -- Dissolve water and make up the volume to 100 ml. 8.1 .lO Sodium Chloride 10 g of sodium 8.1.11 Potassium Iodide Control Solution - Dissolve 0.328 0 g of potassium iodide in water and then make up the volume to 250 ml. Dilute 50 ml of this solution to 250 ml, and use 5-ml control ( that is, 1.0 mg iodine or O-308 mg potassium iodide ). 8.1.12 Concentrated Sulphuric Acidr.d. 1.84 (see IS : 266-1961* ). g of and the the 8.2 Preparation of Sample Solution - Weigh accurately about 50 the material and suspend in 100 ml of water. Add 2 g of takadiastase allow to stand at 37°C for 2 hours. Filter the solution and wash Collect the filtrate and washings and make up residue with water. volume to 250 ml in a~graduated flask. *Specification for sulphuric acid ( revised). 10 IS 17874 -( Part II ) - 1975 8.3 procedure - Pipette 50 ml of the prepared sample solution ( SM 8.2-I ) Neutralize to methyl orange indicator into a 200-ml Erlenmeyer flask. Add bromine water dropwise from burette in a with dilute sulphuric acid. quantity equivalent to 20 mg of bromine. After a few minutes, destroy most of the remaining free bromine by adding sodium sulphite solution dropwise Wash down the neck and sides of the flask with water and with stirring. completely remove free bromine by addition of a drop or two of phenol Add 1 ml of dilute sulphuric acid and 5 ml of potassium iodide solution. solution and titrate the liberated iodine with the standard sodium thiosulphate solution adding 1 ml of the starch indicator near the end of the titration. Carry out a blank determination on reagents and make one or more control determinations, using 50 ml of sodium chloride solution to which have been added appropriate quantities of the potassium iodide control solution. 8.4 Calculation Iodine ( as KI ), on moisture-free basis, percent by mass where vr = volume solution solution, in ml of the standard sodium thiosulphate required for the test with the prepared sample solu= 1 384 ( VI m(lW--M) V,) fl V2 = volume in ml of the standard sodium thiosulphate tion required for the blank determination, _W = normality m = M= percent of the standard moisture content. sodium taken thiosulphate mass in g of the material ( set 13.2 ), and solution, 9. DETERMINATION 9.1 Apparatus 9.1 .O General contamination. 9.1.1 30 ml. 9.1.2 The OF COPPER glassware Tube - used of .50 shall ml be free from and copper at Heat Resistant Glass Centrfuge capable capacity marked of clarifying capable the isoamyl of measuring alcohol phase. density 9.1.3 Photoelectric Calorimeter at 430 nm. 9.2 Reagents 9.2.1 Distilled Water - the optical redistilled. 11 IS : 7874 ( Part II ) - 1975 9.2.2 9.2.3 9.2.4 9.2.5 Sodium Citrate Solution -. saturated. 20 percent Solution ( m/v ). ( m/v ) aqueous. point 129 to 132°C. 0.1 percent Ammonium Hydroxide Solution Isoamyl Alcohol ---boiling Sodium Diethyldithiocarbamate 9.2.6 Standard Copper Solution - Dissolve O-393 g cupric sulphate pentahydrate (CuSOa, 5 H,O ) of analytical grade in distiiled water, add a few drops of concentrated sulphuric acid and make up the volume to one litre in a graduated Aask. Shake well. Pipette out 10 ml of this solution into a one-litre graduated flask and make up the volume, This solution contains 1 pg of copper per millilitre. 9.3 Procedure 9.3.1 Pipette a suitable aliquot of the test solution as prepared in 7.3.1, containing about 10 pg of copper, in the glass tube marked at 30 ml. Add 3 ml of sodium citrate solution and ammonium hydroxide solution until just alkaline to phenolphthalein, followed by 3 ml of ammonium hydroxide solution. Make up the volume to 30 ml with distilled water. Add 10 ml of isoamyl alcohol accurately measured and 1 ml of sodium diethyldithiocarbamate solution. Stopper the tube and shake vigoTransfer enough of the isoamyl alcohol phase meant rously for 20 seconds. for colour measurement to centrifuge tubes and centrifuge for 2 minutes at about 3 000 rev/mm. Measure the absorption of the solution in a suitable photo-electric calorimeter at 430 nm, setting the reading of the blank at zero absorption. The blank is prepared simultaneously by using the same quantities of acid employed in the digestion, making up the volume and developing the colour m the same size aliquot and iu the same manner as in the case of the test solution. 9.5.2 Prepare a series of standards by treating aliquots of the standard cooper solution ( see 9.2.6 ) in the same manner as the test solution, From tlrk absorption of the standard solutions prepare a standard curve plotting absorption values against concentrations. From this curve, obtain the mass of copper present in the test solution and calculate the quantity of copper present in 100 g of the material on moisture-free basis. 19. DETERMINATION 10.1 Apparatus 10.1.1 520 nm. 10.2 Photoelectric Calorimeter capable of measuring optical density of OF MANGANESE Reagents Concentrated Sulphuric Acid r.d. 1.84 ( see IS : 266-1961* ). 10.2.1 *Specification for sulphuric acid ( reviced ). 12 1s : 7874 10.2.2 Sulphurous a cool place. Acidsaturated solution stored (Part II) - 1975 bottle in in an amber 18.2.3 Solution A - Mix 42 ml of water, 2 ml of sulphuric acid, 5 ml of sulphurous acid solution and 1 ml of phosphoric acid. This solution should be freshly prepared before use. 10.2.4 lo.25 10.2.6 10.2.7 Potassium Periodate Solution l-70. r.d. 1.42 ( see IS : 264-1968* ). 10 percent ( m/v ), aqueous. Sodium Metabisulphite Phosphoric Concentrated Acid r.d. Nitric Acid - 10.2.8 Standard Manganese Solution - Dissolve 0.575 6 g of dry potassium permanganate in about 50 ml of water in a beaker of suitable size. Add 40 ml of concentrated sulphuric acid and reduce the permanganate by careful addition of sodium metabisulphite solution until the manganese solution just Oxidize the excess sulphurous acid in the hot solution becomes colourlcss. by the addition of a little nitric acid. Cool and transfer the solution quantitatively to a 2-litre graduated flask. ' Make up the volume and store the solution in a glass-stoppered reagent bottle. This solution contains 0.1 mg of manganese per millilitre. 10.3 Procedure 10.3.1 Weigh accurately about 5 g of the material into a silica dish, char carefully and ash it in a muffle furnace at 600 to 700°C. Cool, extract the ash with 10 ml of solution A ( see 10.2.3 ) for 2 minutes and transfer to a 150-ml beaker. Rinse the dish first with 40 ml of solution A and then with distilled water, collecting the rinsings until the volume is 100 ml. Heat to the boil on a hot-plate and evaporate the solution, using a boiling tube Care should be taken not to allow ~until the volume is reduced to 20 ml. the solution to bump. Allow the solution to stand overnight. Filter through a small disc of ashless filter paper under slight suction into a 150-ml beaker Wash the filter paper and dilute the filtrate with water to about 100 ml. Add 2 ml of phosphoric acid and 0.3 g of potassium periodate. Boil to oxidize the manganese and continue boiling for about 15 minutes after the colour has been apparently fully developed. The final volume should not be less than 50 ml. ( If necessary, boiling water may be added to the solution* while boiling. ) Cool and dilute to 100 ml. Measure the absorption of the solution at 520 nm by means of a suitable photo-electric calorimeter. 10.3.2 Simultatieously carry out a control determination under the same conditions as in 10.3.1 adding 5 ml of the standard manganese solution, 2 ml of sulphuric acid and 2 ml of phosphoric acid to 100 ml of water and oxidizing with potassium periodate as described in 10.3.1. *Specification for nitric acid ( jrst revision ). 13 IS : 7074 ( Part II -) - 1975 10.3.3 Measure the absorption at 520 nm of a series of aliquots of the standard manganese solution ( see 10.2.8 ) treated in the same manner as the test solution. Plot a curve of these absorption values against concentration. From this curve, obtain the mass of manganese in the test solution and calculate the quantity of manganese present in 100 g of the material on moisture-free basis. 11. DETERMINATION 11.1 Apparatus 11.l .l Slpectrophotometer or Photoelectric 11.2 Colorimeter of a suitable type. OF COBALT Reagents * Water redistilled. 11.2.1 Distilled Prepare by dissolving 42 g of citric acid in 11.2.2 Citric Acid - 0.2 M. 100 ml of water and standardize against standard sodium hydroxide solution using phenolphthalein as indicator. 11.2.3 Bromophenol Blue Indicator Solution - Dissolve 40 mg of bromophenoL blue in 100 ml of water containing 5.7 ml of sodium hydroxide ( 0.01 N). 11.2.4 Methyl Red Indicator Solution ml of ethyl alcohol ( 60 percent U/U ). Dissolve 25 mg of methyl 0.2 percent ( m/v ). 0.05 red in 100 Keep in a 11.2.5 Dithizone Solution in Chloroform dark bottle in a refrigerator. 11.2.6 Dithizone Solution in Carbon Keep in a dark bottle in a refrigerator. Tetrachloride percent ( m/v ). 11.2.7 Phenolphthalcin Indicator SolutionDissolve 50 mg of phenolphthalein in 100 ml ethyl alcohol ( 50 percent m/v ). Sorensen's 11.2.9 Solution - Dissolve 6.184 g of boric acid and 25.62 g of salt (NazHP0,,2H20 ) in 500 ml of standard sodium hydroxide ( 1-O N ) and make up the volume to one litre with distilled water. Concentrated ,Nitric Acid 60 percent ( m/v ). 11.2.8 Bujer 11.2.10 Perchloric Acid11.2.11 60 percent. r.d. 1.84 (see IS : 266-1961* ). in Concentrated Sulphuric Acid- 40 mg of cresol red 11.2.12 Cresol Red Indicator Solution -Dissolve 100 ml of water containing 10.5 ml of sodium hydroxide ( O-01 -N ). 11.2.13 .Nitroso-R Salt-O.2 percent (m/v) aqueous solution stored in dark. *Specification for sulphuric acid ( rcvisrd ). 14 IS : 7874 ( Part XI) - 1975 11.2-14 Standard Cobalt Solution -Dissolve 1.769 4 g of cobalt sulphate ( CoSO,, 7H20) in distilled water, add one millilitre of concentrated sulphuric acid and make up the volume to one Iitre. Take one millilitrc of this solution and make up volume to one litre with water in a graduated flask. This solution contains 1 pg of cobalt per millilitre. 11.3 Procedure 11.3.1 Take an aliquot of 5 ml of the test solution as prepared in 7.3.1. Evaporate the water cautiously until all but a trace of sulphuric acid is removed. Add 7.5 ml of nitric acid to the residue and wash the solution into a lOO-ml separating funnel. Dilute to about 30 ml with water. 11.3.2 Extraction u&h Dithimne -- Add 5 drops of bromophenol blue to the solution. Run in sodium hydroxide solution ( 1.0 N ) until a distinct greenish-blue colour appears through the yellowish tint due to the ferric Gitrate. Th e solution should still be acid to methyl red. Dilute the solution to 50 ml. Extract the solution with successive 20 ml portions of dithizonc solution in chloroform. Sh a k e vigorously and run 0-q the chloroform layer. When the chloroform layer retains the original green colour of the dithizone solution, the test solution is washed once with pure chloroform. Adjust thepH of the aqueous phase to approximately 8.3 by adding a few drops of phenolphthalein and cautiously titrating with the buffer Exlract the solution until the first sign of a purplish-pink colour appears. cobalt with successive 10 ml portions of dithizone solution in carbon tetrachlorideu ntil the carbon tetrachloride phase retains the green colour of Boil off the carbon tetrachloride from a the original dithizone solution. heat resistant boiling tuhe. Add to the residue 1 ml of nitric acid, 0.5 ml of perchloric acid and 02 ml of sulphuric acid and heat till it becomes Heat the boiling tube for a few minutes in a muffle furnace at colourless. a temperature not above 350°C to ensure complete removal of sulphuric acid. 11.3.3 Production oj- the Cob&t-Nitroso- R Salt Complex - Dissolve the residue in 1 ml of citric acid and dilute with a little water so that the total volume is not more than5 ml. Add accurately 1.2 ml of the buffer solution The pH is. checked with cresol red by withdrawing a to adjust the PH. Develop the cobalt-nitroso-R salt complex by small drop of the solution. introducing O-5 ml of the cobalt-nitroso-R salt solution while shaking. Boil for one minute. Cool and dilute to 10 ml. Measure the absorption of the solution in a suitable spectrophotometer or photoelectric calorimeter at 510 nm setting the reading of the blank at zero absorption. The blank is prepared simultaneously by using the same quantities of the reagents Make up the employed in the digestion and in the subsequent procedure. volume to 100 ml and develop the colour in the same size aliquot and in the same manner as in the case of the test solution. 15 IS : 7874 ( Part II ) - 1975 11.3.4 Prepare a series of standards by treating aliquots of the standard cobalt solution in the~same manner as the test solution. From the absorption of the standard solutions, prepare a standard curve plotting absorption values against concentrations. From this curve, obtain the mass of cobalt present in the test solution and calculate the quantity of cobalt present in 100 g of the material on moisture-free basis. 12. DETERMINATION OF FLUORINE 12 .l Apparatus 12.1 .l Distillation Flasks 12.1.2 12.1.3 12.2 JVesslerTubes Microburette of 50 ml capacity. Reagents 12.2.1 Lime Water Freed from Fluorine - Dissolve lime in an excess of pcrchloric acid. Boil for 15 minutes. Dilute, cool and neutralize with ilourine-free sodium hydroxide. Filter through a Buchner funnel and wash. solution in distilled water using the lime thus freed from h;l ake a saturated fluorine. 12.2.2 quantity Perchloric Acid Solution - 60 to 70 percent for an hour or longer at 140 to 150°C. ( m/v). Heat some Prepare by adding 22.2.3 Silver Perchlorate Solution - 1 percent ( m/v ). sunicient sodium hydroxide solution to a solution of silver nitrate to cause precipitation. Filter and wash the precipitate with water. Dissolve the prciipitatc in perchloric acid and dilute. 12.2.4 Sodium Hydroxide Solution 0.5 N. alizarin 12.2.5 Alirarzn Itzdicator Solution - Dissolve 0.02 g of sodium sulphonate in water and make up the volume to 100 ml. 12.2.6 Dilute Hydrochloric Acid 0.05 N. 12.2.7 Bufer Solution - Dissolve 0.1 g of hydroxylamine in water and make up the volume to 100 ml. hydrochloride 12.2.8 Thorium Nitrate Solution - Dissolve 0.5 g of hydrated thorium nitrate [Th( NOs )a,12 HsO ] in distilled water and make up the volume to one litre. 12.2.9 Standard Fluorine Solution - Dissolve 2.2 11 g of sodium fluoride in Pipette water and make `up the volume to one litre in a graduated flask. out 10 ml of this solution into a one-litregraduated flask and make up the This solution contains O+Ol mg of fluorine per millilitre. volume. 16 IS : 7874 ( Part II ) - 1975 12.3 Procedure 12.3.1 Weigh accurately about 5 g of the material. Moisten with lime Dry on a water-bath and ignite in a mufIle furnace at about water. 550 f 20°C. When the ashing is complete, cool and transfer to distillation flask, washing it with water. Dissolve the residual ash in 10 to 15 ml of perchloric acid and transfer to the same distillation flask. Add sufficient silver perchlorate to bring about complete precipitation. Steam-distil at 132 & 3°C into another flask containing 2 ml of the sodium hydroxide solution. Collect about 150 ml of the distillate. Transfer the distillate to a 200-ml graduated flask and make up the volume to the mark with water. 12.3.2 Transfer a suitable aliquot of the test solution containing 10 to Add 1 ml of alizarin indicator solution. 30 pg of fluorine to a Nessler tube. Take the same quantity of the indicator solution in another Nessler tube. If necessary, the alkali in the test solution is neutralized with a drop or two of hydrochloric acid. To each tube, add I ml of the buffer solution, 2-O ml of hydrochloric acid and dilute to about 45 ml. The colour of the Add the thorium nitrate solution from a solutions should be straw-yellow. microburette to the test solution unt.il a permanent slight pink colour appears ( 0.5 to 2.5 ml of thorium nitrate solution would be required ). Add an equal volume of the thorium nitrate to the other Nessler tube. Adjust the colour of the solution in this Nessler tube by adding the standard fluorine solution from a micro-burette to the same intensity as the colour of the test solution. When the colour in both the tubes matches, the amount of flourine in the standard Aourine solution added is equal to the amount of flourine present in the aliquot of the test solution. From this, calculate the amount of fluorine present in 100 g of the material on moisture-free basis. 13. DETERMINATION OF ZINC 13.1 Apparatus 13.l .1 Calorimeter photometer 13.2 capable A photo-electric calorimeter or suitable spectroof measuring optical density at a wavelength of 540 nm. Reagents 13.2.1 Copper Sulphatt ( CuSO,, sulphate Solution -Prepared by dissolving 8.0 g copper 5HsO ) in distilled water and diluted to one litre. 13.2.2 Ammonium Citrate Solution - Dissolve 225 g ammonium citrate to phenol red I: ( NH, )s GH@, 3 in distilled water, make alkaline (pH -& 7.4 ) with concentrated ammonium hydroxide ( &25 percent ) and add a further 75 ml. Dilute to 2 litres. Before use, purify by adding a slight excess of dithizone solution ( see 13.2.5 ) and extract with successive portions of carbon tctrachloride until the solvent layer has a clear bright green colour. Remove the dithizone rrmaining in the solution by means of successive cxtract.ions with chloroform followed by a final extraction with carbon tctrachloride. ( l'l or ci i 11 \izonc shall be cntircly removed to prevent loss of zinc- during the renmval of cobalt. I7 ) IS : 7874 ( Part II ) - 1975 13.2.3 a-.h'itros+fLNaphthol Solution - Dissolve 0.25 g cc-nitroso-pnaphthol in chloroform~and make the volume up to 500 ml with chloroform. 13.2.4 Chloroform -redistilled. Store in an amber bottle. 13.2.5 Dithizone Solutioia 1 Dissolve 0.050 g dithizone in 2 ml of 25 percent ammonia solution and make up to 100 ml with water. Extract with successive portions' of carbon tetrachloride until the solvent layer has clear bright green colour. Discard the solvent and filter the aqueous solution through washed ashless filter paper. 13.2.6 13.2.7 Carbon Standard Tetrachloride Hydrochloric -redistilled. Acid Solution 0.04 N. 13.2.8 Standard zinc Solution - Dissolve 0.500 g of pure granulated zinc in a slight excess of hydrochloric acid. Dilute to 1 litre with double.distilled One millilitre of this solution contains 0.5 mg of zinc. water. 13.2.9 Standard zinc Working Solution-Dilute 10 ml of standard zinc solution ( see 13.2.8 ) with 0.04 N hydrochloric acid to make 1 litre. One millihtre of this solution contains 5 pg of zinc. 13.2.10 containing 13.2.11 Ammonium approximately Hydroxide Solution Acid Solution - An aqueous solution of. ammonia 5 percent ammonia ( m/m ). Concentrated 1 : 6. hydrocloric acid diluted with distilled water in the proportion 13.2.12 Bromine 13.2.13 Water -A Hydrochloric saturated solution of bromine in water. Hydrogen Sulphide 13.2.14 Methyl Red Indicator Solution 100 ml 60 percent ethyl alcohol. Dissolve Dissolve 25 mg 100 mg methyl phenol red in red 13.2.15 Phenol Red Indicator Solution sodium salt in 100 ml`distilled water. 13.3 procedure - Dilute 10 ml of the test solution to about 40 ml. Add 2 drops of methyl red indicator and 1 ml copper sulphate solution and neutralize with ammonia. Add sufficient hydrochloric acid solution ( 13.2.11 ) to bring the concentration of this acid to 0.15 N. The J'JH value of the solution Pass a stream of hydrogen sulphide should now be between 1.9 and 2.1. through the solution until precipitation is complete. Filter through a fine filter paper ( previously washed with hydrochloric acid solution and Wash the precipitate and filter paper with water ) into a 250-ml beaker. with three or four small portions of water, adding the washings to the filtrate. ai1 the solution until all trace of hydrogen sulphide has been removed, add 5 ml of bromine water and continue boiling until free from bromine. Cool, IS : 7874 ( Part JII ) - 1975 neutralize to phenol red with ammonium hydroxide solution and add O-6 ml hydrochloric acid solution. Make up to a suitable volume and take an aliquot containing 4 to 20 pg of zinc for the determination. Adjust the volume of the aliquot to about 20 ml by the addition of distilled water and transfer to a 125-ml separating funnel. Add 5 ml ammonium citrate solution and l0 ml a-nitroso+-naphthol solution. Shake for 2 minutes, allow the phases to separate and discard the solvent layer. 13.3.1 Wash the aqueous layer with small portions of chloroform to remove residual cr-nitroso-P-naphthol. If necessary, adjust the #H value of the aqueous solution to 8.0 to 8.2 by the addition of ammonium hydroxide or hydrochloric acid solutions and add 2 ml dithizone solution and 10 ml carbon tetrachloride. Shake for 2 minutes, allow the phases to separate and, using a pipette, withdraw the aqueous phase as completely as possible and discard it. Wash down the sides of the separating funnel. with 25 ml distilled water and again withdraw the aqueous phase land discard it. Add 25 ml of 0.04 N hydrochloric acid to the contents of the separating funnel, shake for 1 minute to transfer zinc to the aqueous phase, allow the phases to separate and discard the solvent layer. To the aqueous solution'remaining in the separating funnel add 5 ml ammonium citrate solution and adjust the PH value, if necessary, to 8.8 to 9.0. Add IO.0 ml carbon tetrachloride accurately measured. Determine the quantity of dithizone solution to be added as follows. 13.3.1.1 To a separating funnel containing 4.0 ml of the standard zinc working solution ( 20 pg zinc ) made up to 25 ml with O-04 N hydrochloric acid ad3 5.0 ml ammonium citrate solution and 10 ml carbon tetrachloride; then add dithizone solution from a burette in 0.1 ml increments, shaking after each addition, until a faint yellow colour in the aqueous phase indicates a slight excess of reagent. Multiply the volume of dithizone solution used by 1.5 and add this quantity to the test solution. Shake for 2 minutes and allow the phases to separate. Pipette 5 ml of the solvent phase into a test tube, dilute with 10 ml carbon tetrachloride and use for the determination of the absorption. Prepare a series of standards containing 0.5, 10, 15 and 20 pg zinc diluted in each case to 25 ml with 0.04 N hydrochloric acid solution and treated in the same manner as the test solution. Prepare a blank simultaneously by using the same quantities of reagents as were used in the digestion of the test sample and in the subsequent procedure, making up to 200 ml and developing the colour in the same size aliquot and in the same manner as in the actual determination. Measure the absorptions of the standard and test solutions at 540 nm ill Prom the absorptions of the standard solutions pr'p_ul. ;s the calorimeter. graph by plotting absorptions ag:Linst Co:1~.Cllf.l~\t.i~~lls ;uld ir